The growth of Brettanomyces bruxellensis is a major cause of wine spoilage due tothe production of the volatile phenols 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG), derivatives of p-coumaric acid and ferulic acid. During the winemaking process,some microorganisms can impact the concentration of the 4-EP precursors pcoumaricand coutaric acid (tartaric acid-esterified p-coumaric acid). This studyinvestigated the effect of certain wine microorganisms on these 4-EP precursors aswell as potential inhibitory relationships between B. bruxellensis and the winebacteria Oenococcus oeni. Saccharomyces cerevisiae strains with and withoutphenolic acid decarboxylase (PAD) activity as well as Oenococcus oeni strains withand without cinnamoyl esterase (CE) activity were used to perform sequential andsimultaneous Pinot noir alcoholic fermentations (AF) and malolactic fermentations(MLF). Simultaneous fermentation using PAD (+) S. cerevisiae and a CE (+) O. oeniresulted in the largest reduction of volatile phenol precursors. However, these samestrains when utilized in sequential fermentations resulted in the largest number ofprecursors in the form most readily usable by B. bruxellensis. Several commercial and non-commercial O. oeni were also screened for CE activity. Two of the elevenpreviously unscreened commercial O. oeni as well as one of the four non-commercialO. oeni exhibited cinnamoyl esterase activity, resulting in significantly higherconcentrations of free hydroxycinnamic acids in these wines. The stability of the 4-EP precursor compounds during aging was investigated by aging Pinot noir winesthat had undergone MLF using either a CE (+) or CE (-) O. oeni. Wines were adjustedto different pH and ethanol concentrations and stored at either 13 or 21C. During180 days of aging, the concentration of p-coumaric acid and coutaric acid remainedrelatively stable and were most impacted by the strain of O. oeni that had conductedMLF.While O. oeni can impact B. bruxellensis wine spoilage through the liberation oftartaric acid-bound hydroxycinnamic acids, it has also been noted that the presence ofO. oeni at the completion of MLF may inhibit B. bruxellensis growth. Experimentswere conducted to determine how long this inhibition lasted as well the sensitivity ofmultiple B. bruxellensis strains. Three different O. oeni strains were used to conductMLF in a Pinot noir wine. Upon completion of MLF, wines were pulled andinoculated with B. bruxellensis at 0, 34, and 112 days post-MLF. Growth of B.bruxellensis and O. oeni were monitored and volatile phenol (4-ethylphenol and 4-ethylguaiacol) concentrations in the wine were also measured. O. oeni populations inthe wines were high directly after the completion of MLF and B. bruxellensispopulations declined rapidly following inoculation. 34 days post-MLF, highpopulations of two O. oeni strains were still present in the wine but no culturable cells were detected in the wine for the third strain. When B. bruxellensis was inoculatedinto the wine containing no culturable O. oeni cells, it grew well. In contrast, B.bruxellensis populations rapidly declined when inoculated into wine where there werestill culturable O. oeni cells. 112 days post-MLF, no culturable O. oeni cells weredetected in any of the wines and B. bruxellensis grew well in all but one of the wines.The sensitivity of different B. bruxellensis strains to the presence of culturable O.oeni post-MLF was tested by inoculating six strains of B. bruxellensis into wine thathad undergone MLF by O. oeni or had not (control). Subsequent growth and volatilephenol production was tracked. The degree of inhibition on growth and volatilephenol production was strain dependent where the growth of some B. bruxellensisstrains in wine that underwent MLF was comparable to growth in the control wines.For other strains, there was reduced or no growth in wines that had undergone MLF.All six strains of B. bruxellensis produced significantly less volatile phenols in winesthat had undergone MLF compared to those that had not. These findings suggest thatMLF may offer limited protection against B. bruxellensis infection due to thepresence of live O. oeni cells post-MLF but that this may be dependent on the B.bruxellensis strain present. Additional research should involve screening a largernumber of B. bruxellensis strains for their sensitivity to live O. oeni cells. Focusingon B. bruxellensis strains which have been sequenced and better classified from an-omics point of view would likely provide better insight behind variations ininhibition as well as possible mechanisms of inhibition.
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